1. Field of the Invention
The present invention relates to methods and compositions for the fermentative production of ethanol from D-xylose using yeast.
2. Background
Metabolic engineering of microorganisms is often an effective means to produce commercially a number of chemicals that may be used for multiple applications (see, e.g., Lee, S. Y., et al. Macromol. Biosci. 4:157-164 (2004)). One chemical that has garnered much interest is ethanol. Although ethanol has a number of uses, it is most commonly used as a fuel additive. As a fuel additive, ethanol is a low value product with much of the cost of its production attributed to the cost of raw materials. It would be desirable, therefore, to develop ethanologens and fermentation processes for the production of ethanol from readily available, inexpensive starting materials. These starting materials may be, for example, lignocellulosics. These lignocellulosics may be derived from renewable biomass waste streams from food, paper pulping operations, agricultural residues and recycled paper from municipalities.
The major constituent of plant biomass is lignocellulose. Upon hydrolysis, lignocellulose yields a mixture of monomeric hexoses (glucose, mannose and galactose) and pentoses (D-xylose and L-arabinose). Among these, glucose is the most abundant, followed by xylose and mannose with other sugars present in much lower concentrations. Fermentation of both glucose and xylose is currently regarded as a high priority for economical conversion of biomass into ethanol. Most microorganisms are able to ferment glucose but few have been reported to utilize xylose efficiently and even fewer ferment this pentose to ethanol. However, the competitive process for fuel ethanol production from lignocellulosic material requires the development of microbes capable of active xylose fermentation.
Lignocellulose is approximately 30% D-xylose (see Ryabova, O. B., et al. “Xylose and Cellobiose Fermentation to Ethanol by the Thermotolerant Methylotrophic Yeast Hansenula polymorpha,” FEMS Yeast Res. 4:157-164 (2003)). Xylose is a “wood sugar” with the IUPAC designation (2S,3R,4S,5R)-oxane-2,3,4,5-tetrol.
Only a relatively small number of wild type microorganisms can ferment D-xylose. These microorganisms are generally not suitable for large-scale fermentation. This unfavorability may arise, for example, as a result of unfamiliarity with the microorganisms, difficulty obtaining the microorganisms, poor productivity and/or growth on pretreated lignocellulosics or unsatisfactory yield when grown on mixed sugars derived from biomass.(C. Abbas, “Lignocellulosics to ethanol: meeting ethanol demand in the future,” The Alcohol Textbook, 4th Edition. (K. A. Jacques, T. P. Lyons and D. R. Kelsall, eds). Nottingham University Press, Nottingham, UK, 2003, pp. 41-57.; C. Abbas, “Emerging biorefineries and biotechnological applications of nonconventional yeast: now and in the future,” The Alcohol Textbook, 4th Edition. (K. A. Jacques, T. P. Lyons and D. R. Kelsall, eds). Nottingham University Press, Nottingham, United Kingdom, 2003, pp. 171-191).
Yeasts are considered the most promising microorganisms for alcoholic fermentation of xylose (see Ryabova, supra). They have larger cells than bacteria, are more resistant to viral infection, and tend to be more resistant to negative feedback from ethanol. Furthermore, yeast growth and metabolism have been extensively studied for a number of species.
A number of yeasts are known to naturally ferment D-xylose. These include Pichia stipitis, Candida shehatae, and Pachysolen tannophilus (see Ryabova, supra; Cite 2, C. Abbas 2003). The common brewer's yeast Saccharomyces cerevisiae is not known to ferment D-xylose naturally, but a number of strains of metabolically engineered S. cerevisiae that do ferment D-xylose have been reported.
Numerous studies have described the metabolism of D-xylose by recombinant S. cerevisiae (see, e.g., Wahlbom, et al., “Metabolic Engineering for Improved Xylose Utilization of Saccharomyces Cerevisiae,” U.S. Pat. Pub. No. 2005/0153411A1 (Jul. 14, 2005); Griffin, et al., “Method of Processing Lignocellulosic Feedstock for Enhanced Xylose and Ethanol Production,” U.S. Pat. Pub. No. 2004/0231661A1 (Nov. 25, 2004); Gong, C-S, “Direct Fermentation of D-Xylose to Ethanol by a Xylose-Fermenting Yeast Mutant,” U.S. Pat. No. 4,368,268 (Jan. 11, 1983); Hallbom, J., et al., “Production of Ethanol from Xylose” U.S. Pat. No. 6,582,944 (Jun. 24, 2003); Jeffries, T. W, et al., “Xylose-Fermenting Recombinant Yeast Strains,” U.S. Pat. Pub. No. 2004/0142456A1 (Jul. 22, 2004); Jeffries, T. W. & Jin, Y-S., “Metabolic Engineering for Improved Fermentation of Pentoses by Yeasts” Appl. Microbiol. Biotechnol. 63: 495-509 (2004); Jin, Y-S. & Jeffries, T. W., “Stoichiometric Network Constraints on Xylose Metabolism by Recombinant Saccharomyces cerevisiae” Met. Eng. 6: 229-238 (2004); Pitkanen, J-Y., “Impact of Xylose and Mannose on Central Metabolism of Yeast Saccharomyces cerevisiae” Helsinki Univ. of Tech., Dept. of Chem. Tech., Technical Biochemistry Report (January 2005); Porro, D., et al., “Replacement of a Metabolic Pathway for Large-Scale Production of Lactic Acid from Engineered Yeasts” App. & Env. Microbiol. 65(9): 4211-4215 (1999); Jin, Y-S., et al., “Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response” App. & Env. Microbiol. 70(11): 6816-6825 (2004); Sybirna, K, et al., “A New Hansenula polymorpha HAP4 Homologue which Contains Only the N-Terminal Conserved Domain of the Protein is Fully Functional Saccharomyces cerevisiae” Curr. Genetics 47(3): 172-181 (2005); Toivari, M. H., et al., “Conversion of Xylose to Ethanol by recombinant Saccharomyces cerevisiae: Importance of Xylulokinase (XKS1) and Oxygen Availability” Metabolic Eng. 3:236-249 (2001).
As shown in FIG. 1, D-Xylose metabolism in yeast proceeds along a pathway similar to that of glucose via pentose phosphate pathway. Carbon from D-xylose is processed to ethanol via the glycolytic cycle or to CO2 via respiratory TCA cycle.
Fermentation to ethanol relies in part on the metabolism of pyruvate, which is a metabolite that may be used in either respiration or fermentation (see van Hoek, P., et al., “Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in Saccharomyces cerevisiae,” Appl. & Enviro. Microbiol. 64(6); 2133-2140 (1998)). Pyruvate enters fermentation following decarboxylation of pyruvate to acetaldehyde by the enzyme pyruvate decarboxylase (E.C. 4.1.1.1). Pyruvate decarboxylase is a member of the family of biotin-dependent carboxylases. It catalyzes the decarboxylation of pyruvate to form oxaloacetate with ATP cleavage. The oxaloacetate can be used for synthesis of fat, glucose, and some amino acids or other derivatives. The enzyme is highly conserved and found in a variety of prokaryotes and eukaryotes.
Pyruvate decarboxylase was first reported by (Utter, M. F., et al., “Formation of oxaloacetate from pyruvate and CO2” J. Biol. Chem. 235:17-18 (1960)) while defining the gluconeogenic pathway in chicken liver. Attempts to overexpress the PDC1 gene in S. cerevisiae did not resulted in higher ethanol yield from glucose (Schaaff I, Heinisch J & Zimmermann F K (1989) “Overproduction of glycolytic enzymes in yeast.” Yeast 5(4): 285-290; van Hoek P, Flikweert M T, van der Aart Q J, Steensma H Y, van Dijken J P & Pronk J T (1998) “Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch point in Saccharomyces cerevisiae.” Appl Environ Microbiol 64(6): 2133-2140.)
It has been proposed that one bottleneck involved in D-xylose fermentation is the hydrolysis of xylan, which is the major component of hemicellulose to monosaccharides (see Ryabova, supra). One approach to overcoming this bottleneck is by using “simultaneous saccharification and fermentation” (SSF). This is a process in which pretreated lignocellulose is hydrolyzed by cellulases and hemicellulases while the hexoses and pentoses produced by this hydrolysis (including xylose) are fermented to ethanol. This would allow continuous conversion of the sugars to ethanol, preventing their accumulation in the medium.
A drawback of SSF is the difference in the optimal temperature at which cellulases and hemicellulases are active (at least about 50° C.) that are compatible with the optimal temperature for yeast growth and fermentation of xylose (about 30° C.). One solution to this drawback is to perform SSF using the thermotolerant methylotrophic yeast Hansenula polymorpha (also known as Pichia angusta). This yeast has been reported to have optimum and maximum growth temperatures of 37° C. and 48° C., respectively. These temperatures are higher than those tolerated by most other ethanol producing yeasts (Ryabova, et al.). Furthermore, Ryabova, et al. reported that under some conditions H. polymorpha is able to naturally ferment D-xylose (see also Voronovsky, A. Y., et al., “Expression of xylA Genes Encoding Xylose Isomerases From Escherichia coli and Streptomyces coelicolor in the Methylotrophic Yeast Hansenula polymorpha” FEMS Yeast Res. 5(11): 1055-62 (2005)).
Therefore it would be advantageous to develop strains of H. polymorpha with an increased ability to produce ethanol from lignocellulosic starting materials, including the C5 sugar, D-xylose.